Structural and mechanistic insights into the transport of aristolochic acids and their active metabolites by human serum albumin

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M−1 for HSA/AA-I versus 8.4 and 9.0 · 105 M−1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.


Figure S1 .
Figure S1.HSA-AA Binding Profiles Deduced via Fluorescence Quenching Assays of Trp 214 .HSA A3782 (closed circles) and HSA A8763 (open circles) are incubated in the presence of AA-I, AA-II, AL-I-NOH, AL-II-NOH, AL-I-NOSO 3 , and AL-I-NOAc.The intrinsic Trp 214 fluorescence intensity (λ EX = 295 nm; λ EM = 340 nm) is recorded for each HSA/AA ratio to create the corresponding ligand-induced quenching profile.The maximum fluorescence intensity observed for HSA in the absence of AA species is assigned as reference (100 AU).Each data point represents the mean and standard deviation for three to five independent equilibrium titration experiments.

Figure S2 .
Figure S2.Apparent Dissociation Constants (K D app ) for AA and Metabolite Complexes in the Absence (HSA A3782 ) and Presence (HSA A8763 ) of Fatty Acids.Data are reported as the mean ± SD with statistical significances deduced via One-Way ANOVA and Tukey Tests employing confidence levels of p < 0.10 ( * ) and p < 0.05 ( ** ).Respective differences in K D app are calculated for AA ligand interactions with fat-free (HSA A3782 , solid) relative to fat-containing (HSA A8763 , checkerboard) protein preparations.

Figure S3 .
Figure S3.Structure of the HSA MYR Subdomain IB Binding Site and Corresponding Key Amino Acids.The subdomain IB binding cleft adopts an "open" conformation and is occupied by two distinct myristate molecules.One myristate ligand is bound at the typical FA1 position (cyan) deep within the binding site.An additional myristate molecule (FA10) is located at a secondary binding position near the lip of site IB (salmon) in the vicinity of H146.

Figure S4 .
Figure S4.Aristolochic Acid I Bound in the HSA Subdomain IB Site.A: AA-I binds deeply in subdomain IB at the original location of FA1 with only the carboxylate and nitro groups protruding towards solution.B: Shape of the subdomain IB binding pocket viewed via cutaway of the HSA molecular surface.AA-I is bound proximate to the inner side where it is surrounded by aliphatic and aromatic residues.The carboxylate and nitro groups point towards a cluster of positively charged resides (e.g., R117, R186) at the "lip" of this pocket.

Figure S5 .
Figure S5.Fo-Fc and 2Fo-Fc Electron Density Maps of AA-I and AA-II Bound in Subdomains IB and IIA, respectively.The Fo-Fc (green mesh) (A) and 2Fo-Fc (blue mesh) (B) electron density maps of AA-I bound in subdomain IB contoured at 3σ and 1σ, respectively.The Fo-Fc (green mesh) (C) and 2Fo-Fc (blue mesh) (D) electron density maps of AA-II bound in subdomain IIA contoured at 3σ and 1.5σ, respectively.

Figure S6 .
Figure S6.Ligplot Diagrams Depicting Binding Sites in HSA/AA-I (A) and HSA/AA-II (B, C).A: Interactions formed between AA-I and residues of the subdomain IB pocket in the HSA MYR /AA-I complex B: Interactions formed between AA-II and residues of subdomain IIA (Drug Site I) in the HSA MYR /AA-II complex C: Interactions formed by AA-II with the lip region of site IB in the HSA MYR /AA-II complex.Hydrogen bonds are represented by green dashed lines and hydrophobic contacts by red spokes.

Figure S7 .
Figure S7.Aristolochic Acid II Bound to HSA Subdomain IIA (Drug Site I).A: AA-II is bound within Drug Site I located in subdomain IIA near the interface between subdomains IB, IIA, and IIIA.Consistent with AA-I, the narrow solvent channel reveals that AA-II is buried within the binding site.B: The general shape of Drug Site I and AA-II bound therein viewed via cutaway of the HSA molecular surface.Most of the subdomain IIA pocket is occupied by AA-II with its aromatic section positioned towards the lipophilic region.The carboxylate and nitro groups of AA-II protrude towards two narrow solvent channels and interact with nearby basic residues.

Figure S8 .
Figure S8.Aristolochic Acid II and FA1 Myristate Bound in HSA Subdomain IB.A: AA-II (orange spheres) and myristate FA1 (cyan spheres) occupy the binding pocket with their carboxylate and nitro groups facing the solvent exposed entrance.B: A cutaway view of the HSA molecular surface exposing specific interactions at the subdomain IB binding site.AA-II is bound at the lip of this pocket and a myristate molecule occupies the FA1 position.Both AA-II and FA1 are secured by hydrophobic interactions within the pocket as well as hydrogen bonding and electrostatic interactions at the lip of this binding site.

Figure S9 .
Figure S9.Superimposition of Aristolochic Acids I (A) and II (B) with Ligands that Commonly Bind to HSA Subdomains IB and IIA.A: AA-I (orange) and hemin (light blue) bound in HSA subdomain IB (Drug Site III).Both ligands bind deeply within the subdomain IB pocket as their planar portions engage in hydrophobic and π-stacking interactions while the complex is stabilized by hydrogen bonding and electrostatic interactions with residues at the mouth of this site.B: AAII (orange) and warfarin R-(+) (PDB: 1H9Z) (blue) bound in HSA subdomain IIA (Drug Site I).The cyclic moieties of both ligands occupy a similar plane within subdomain IIA that positions their hydrophilic groups in an orientation facilitating hydrogen bonding to basic residues at the entrance of this binding site.

Figure S11 .
Figure S11.Crystals of HSA, Aristolochic Acids, and HSA/AA Complexes.A: Typical crystals of the HSA MYR /AA-I complex with dimensions on the order of 0.2 x 0.25 x 0.4 mm and a distinct yellow coloration reflecting bound AA-I.B: Minute metabolite crystals in an HSA MYR /AL-NOH co-crystallization drop.C: Concurrent growth of small rectangular HSA crystals and dark orange metabolite needles under HSA DEFATTED /AL-NOH co-crystallization conditions.

Table S1 . Apparent Dissociation Constants (K Dapp ) Deduced via Fluorescence Quenching Assays of HSA by Aristolochic Acids and Metabolites.
The table compiles individual B-factors and occupancies of ligands present in the crystal structures.Per-ligand averages appear in the two right most columns while per-chain Fatty Acid and Aristolochic Acid I/II averages are listed in the two bottom lines.

Table S2 . Ligand B-factors and Occupancies.
Inspection of the HSA MYR /AA-I and HSA MYR /AA-II complexes reveals that buried solvent accessible surface area exceeds 90 percent for the AA ligands bound to subdomains IB (AA-I/AA-II) and IIA (AA-II).